In this study, polydimethylsiloxane/polyacrylonitrile/titanium dioxide (PDMS/PAN/TiO2) nanofibers were synthesized via electrospinning method to extract and quantify alpha-linolenic acid (ALA, C18:3), as a model analyte, in milk by direct immersion-solid phase nanoextraction (DI-SPNE) with gas chromatography-flame ionization detector (GC-FID). The affecting factors on the electrospinning process such as, PDMS concentration, amount of TiO2 nanoparticles (NPs), voltage, and electrospinning distance were optimized using Taguchi's orthogonal design. The SPNE experimental conditions such as, extraction time, agitation rate, pH and salt concentration, were also optimized using response surface methodology (RSM) based on a central composite design (CCD). Under optimized conditions, the resulting calibration curve was linear in the range of 1-4000 ng with R-2 = 0.998. The intraday, interday, and fiber-to-fiber repeatability were calculated and the corresponding relative standard deviation was less than 9% in all the cases. The limit of detection and limit of quantification were found to be 0.2 and 0.6 ng mL(-1), respectively. Omega-3 enriched milk was used as a real sample and the value of relative recoveries were measured to be in the range of 92-106%.